The ability to isolate low molecular weight nucleic acid, and extrachromosomal nucleic acid in particular, from a host cell is often requisite to a large number of protocols used in molecular biology, as well as a basic requirement in a number of downstream uses in biotechnology and clinical research. For example, typical cloning protocols anticipate the availability of plasmid vector DNA for the transformation of target cells. The quality, i.e., level of purity and integrity, of the extrachromosomal nucleic acid is often determinative of the success of the cloning procedure, and as such, is a critical parameter for the entire procedure. Further, DNA sequencing, restriction digestion reactions and subsequent ligation reactions, are generally dependent on the quality of the starting DNA material. As such, there has been, and continues to be, a need for reliable methods for purifying high quality low molecular weight nucleic acid from host cells.
Conventional low molecular weight nucleic acid purification schemes often progress in more or less two stages: in the first stage, host cells harboring the target nucleic acid, i.e. extrachromosomal nucleic acid, are gently lysed and the contents solubilized; and in the second stage, the target nucleic acid is separated from the contaminating protein, RNA, high molecular weight nucleic acid (i.e. chromosomal DNA), and other macromolecules via one of several commonly used chemical or enzymatic methods. In general, conventional target nucleic acid purification schemes have proven to be either labor intensive and time consuming, yet yielding a high quality product, or relatively fast and labor efficient, but yielding a relatively low quality product.
More particularly, one of the more commonly used time efficient methods for isolating target nucleic acid involves an alkaline lysis technique. The alkaline lysis method typically incorporates a NaOH/SDS lysis solution in sequential combination with a potassium acetate solution, and centrifugation steps to preferentially release and separate the target nucleic acid from other contaminating materials. A separate centrifugation or filtration step is used to produce a cleared lysate. Alcohol precipitation of the cleared lysate is necessary to precipitate the nucleic acid. Although the alkaline lysis method is fairly rapid, it takes approximately 30 to 45 minutes, and the purity of the resultant nucleic acid is fair, i.e., useful in restriction digestions and other more basic detection type procedures, it does not provide quality extrachromosomal nucleic acid.
In a further approach, the cleared lysate as prepared by the alkaline lysis procedure can be combined with a chaotropic substance, for example guanidinium salt, urea and sodium iodide, in the presence of a DNA-binding solid phase (e.g. beads or other binding matrix) to purify the target nucleic acid. The nucleic acid is bound to the solid phase in a one-step reaction, washed to remove residual contaminants and the nucleic acid is then eluted in low salt buffer. Although methods combining alkaline lysis with the chaotropic bind/wash/elute steps can provide higher quality nucleic acid, they are still time-consuming, taking approximately twenty-five minutes, and require more handing.
As such, there is a continuing need in the art for a simple and time efficient method, and corresponding solutions, for purifying a low molecular weight target nucleic acid such as extrachromosomal DNA from host cells, and in particular, for methods and solutions for purifying plasmid DNA from host cells. Against this backdrop the present invention has been developed.